Ecotoxicology and Environmental Safety 113 (2015) 287–294

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Ecotoxicology and Environmental Safety

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Biochemical changes in certain enzymes of Lysapsus limellium(Anura: Hylidae) exposed to chlorpyrifos

Andrés Maximiliano Attademo a,b,n, Paola Mariela Peltzer a,b, Rafael Carlos Lajmanovich a,b,Mariana Cabagna-Zenklusen a,d, Celina María Junges a,b, Eduardo Lorenzatti c,Carolina Aró d, Paula Grenón a

a Ecotoxicology Laboratory, Faculty of Biochemistry and Biological Sciences, National University of Littoral (ESS-FBCB-UNL), Paraje el Pozo s/n, 3000 Santa Fe,Argentinab National Council for Scientific and Technical Research (CONICET)-Faculty of Biochemistry and Biological Sciences (ESS-FBCB-UNL), Paraje el Pozo s/n, 3000Santa Fe, Argentinac Contamination and Remediation soil Signature. Faculty of Biochemistry and Biological Sciences, National University of Litoral (ESS-FBCB-UNL), Paraje elPozo s/n, 3000 Santa Fe, Argentinad Normal Morphology signature -Faculty of Biochemistry and Biological Sciences (ESS-FBCB-UNL), Paraje el Pozo s/n, 3000 Santa Fe, Argentina

a r t i c l e i n f o

Article history:Received 9 June 2014Received in revised form1 December 2014Accepted 9 December 2014

Keywords:Rice fieldLysapsus limellumBiomarkersChlorpyrifos

x.doi.org/10.1016/j.ecoenv.2014.12.02113/& 2014 Elsevier Inc. All rights reserved.

esponding autor at: Ecotoxicology Laboratoryal Sciences, National University of Littoral (ESS) Santa Fe, Argentina.ail address: mattademo@hotmail.com (A. Max

a b s t r a c t

Different enzyme biomarkers (AChE: acetylcholinesterase, CbE: carboxylesterase, GST: glutathione-S-transferase, CAT: catalase) were measured in digestive tissues of Lysapsus limellum frogs collected from arice field (RF: chlorpyriphos sprayed by aircraft) and a non-contaminated area (RS: reference site), im-mediately (24 h) and 168 h after aerial spraying with chlorpyrifos (CPF). CPF degradation was alsosearched in water samples collected from RF and RS, and found that insecticide concentration was re-duced toE6.78% of the original concentration in RF at 168 h. A significant reduction of AChE and CbEactivities was detected in L. limellum from RF in stomach and liver at 24 and 168 h, and in intestine onlyat 24 h, with respect to RS individuals. CAT activity decreased in intestine of L. limellum from RF 24 h and168 h after exposure to CPF, whereas GST decreased in that tissue only at 24 h. In stomach and liver, adecrease was observed only at 168 h in both CAT and GST. The use of biomarkers (AChE, CbE, GST, andCAT) provides different lines of evidences for ecotoxicological risk assessment of wild frog populations atsites contaminated with pesticides.

& 2014 Elsevier Inc. All rights reserved.

1. Introduction

The expansion of rice fields is one of the anthropogenic activ-ities associated with the disappearance of wetlands and lenticenvironments throughout the world (Machado and Maltchik,2010). Despite landscape simplification, rice fields present abun-dance of certain aquatic species that can adjust their biological andecological traits to this homogeneous environment, a fact that wasespecially demonstrated for anuran amphibians (Bambaradeniyaet al., 2004; Duré et al., 2008).

In Argentina, rice is a highly valuable crop cultivated undersubmerged conditions at the expense of considerable freshwaterresources and pesticides (Tilman et al., 2002). Organophosphorus

, Faculty of Biochemistry and-FBCB-UNL), Paraje el Pozo s/

imiliano Attademo).

pesticides (OP) are an important group of agrochemicals widelyused in this type of agroecosystem. OP are rapidly metabolized invertebrate and invertebrate organisms (Racke, 1992) and have arelatively short persistence in the environment, in the order ofhours to few days (Lacorte et al., 1995), with hydrolysis, photolysisand microbial degradation being the main destruction mechan-isms of these ephemeral molecules.

Several biomarkers are increasingly included in environmentalmanagement to assess the health of organisms in complex eco-systems (Galloway et al., 2002). Biomarkers have the advantage ofdetecting early adverse effects resulting from exposure to pesti-cides and altered physical–chemical parameters (oxygen, pH, andnutrients). Consequently, a suite of biomarkers, such as measur-able endpoints at molecular, cellular and physiological levels, maybe necessary to successfully determine the health status of po-pulations, and once selected for a particular case, they can beadapted to different ecological scenarios (Sánchez–Hernández,2007; Almeida et al., 2010). OP pesticides exert acute toxicity by

A. Maximiliano Attademo et al. / Ecotoxicology and Environmental Safety 113 (2015) 287–294288

inhibiting B-esterase enzymes such as acetylcholinesterase (AChE,EC: 3.1.1.7) in the neuromuscular function; in cases of severe in-toxication, OP provoke seizures and respiratory failure, causing thedeath of biota (Fukuto, 1990). However, OP detoxification ismediated primarily by carboxylesterases (CbEs, EC 3.1.1.1; Sogorband Vilanova, 2002; Wheelock et al., 2008). Thus, B-esterase in-hibition by OP in tissues may persist for days to weeks (Morganet al., 1990; Wijeyaratne and Pathiratne, 2006), even if they nolonger contain detectable traces of pesticides.

Glutathione S-transferases (GST EC 2.5.1.18) are another groupof biotransformation enzymes that has been widely used as en-vironmental biomarker. These enzymes catalyze the conjugationof xenobiotics and their metabolites with endogenous glutathioneto facilitate their elimination from the organism (Greulich andPflugmacher, 2004). Another key cellular defense mechanismagainst hydroperoxides that may be the target of pesticide actionis the enzyme catalase (E.C. 1.1.1.6, CAT). CAT activity has beenanalyzed in different organisms exposed to diverse pollutants,including pesticides (Greulich and Pflugmacher, 2004; Ferrariet al., 2011; Peltzer et al., 2013).

In the present study, differences in levels of enzymatic activityof AChE, CbE (using substrates; a-naphthyl acetate), GST, and CATof Lysapsus limellum frogs present in a rice field were studied andcompared with similar data from individuals sampled from a re-ference site (pristine forest) located in Mid-eastern Argentina. Inaddition, we investigated chlorpyrifos (CPF) degradation in watercollected from each sampling sited. Enzymatic biomarkers areusually used to detect biological effects of exposure to pesticideson amphibian larvae and adults (Robles-Mendoza et al., 2011;Peltzer et al., 2013; Ilizaliturri–Hernández et al., 2013; Attademoet al., 2014a). These endpoints provide important lines of evi-dences in ecotoxicological risk assessments (den Besten et al.,2003). Therefore, this study may be useful for ecotoxicological riskassessment of anurans present in rice agroecosystems.

Fig. 1. Location of sampling sites in Mid-eastern Argentina. RS: refer

2. Material and methods

2.1. Study area

The study was conducted in Santa Fe province, located in Mid-eastern Argentina. Rice productions extending on the floodplainsof Paraná River dominate this area (Alvisio, 1998). The area pre-sents a rainy season that extends from October to March and a dryseason from April to September. Rice fields in this region aresurrounded by few and dispersed forest fragments characterizedby native vegetation of the Deltas and Islands of Paraná River andEspinal Ecoregions (Burkart et al., 1999; Peltzer et al., 2004).

2.2. Fields survey

The effects of realistic CPF exposure on amphibians werecompared between two sites, intensively managed rice field (RF)and a reference site (RS) (Fig. 1). The former (RS) was locatedwithin riparian forests along Paraná River in Garay department,Santa Fe province, Argentina (31°10′21.10″S; 60°15′31.73″W). RFwas a rice (Oryza sativa) cropland located in San Javier department(30° 05′ 13.56″ S-59° 53′ 19.98″ W). On 30 January 2013, the in-secticide formulation (commercial grade; 48% a.i.) of CPF (Termi-nator, AGROS Soluciones, Argentina) was applied by aircraft at aproportion of 500 cm3/ha. After that CPF application, mortality ofL. limellum individuals was detected, reaching approximately 3-4dead frogs per m2. Likewise, dead invertebrates (ants, lobsters, andworms) and different species of fishes were also observed. Mor-tality incidents involving L. limellum and other anurans (Pseudisparadoxa, Leptodactylus chaquensis, Dendropsophus nanus) werepreviously recorded in rice crops (Lajmanovich et al., 2012).

The other site (RS) was a wetland without any agricultural ac-tivities in the nearby areas and previous reports determined thatthis site is free of anticholinesterase chemicals (Lajmanovich et al.,2010; Attademo et al., 2014a,b). RS is dominated by different treespecies, such as Salix humboldtiana, Tessaria integrifolia, En-terolobium contortisiliquum, and Acacia caven. The ephemeral

ence site and RF: rice field subjected to chlorpyrifos application.

Table 1Summary of pesticide residue data of water from a reference site (RS) and a ricefield (RF) subjected to chlorphyrifos application.

Pesticide residues7SE (ng L�1) RS RF

24 h 168 h 24 h 168 h

Chlorpyrifos ND(10) ND(10) 138870.2 97.075.1γ-HCH (Lindane) ND (1) ND(1) ND(1) ND(1)

Aldrin ND(2) ND(2) ND(2) ND(2)

Dieldrin ND(8) ND(8) ND(8) ND(8)

p,p-DDT y o,p-DDT ND(10) ND(10) ND(10) ND(10)

p,p-DDE y p,p-DDD ND(8) ND(8) ND(8) ND(8)

Heptachlor epoxide ND(2) ND(2) ND(2) ND(2)

γ and α-Chlordane ND(4) ND(4) ND(4) ND(4)

Heptachlor ND(2) ND(2) ND(2) ND(2)

Endosulfan I and II ND(4) ND(4) ND(4) ND(4)

Endosulfan sulfate ND(4) ND(4) ND(4) ND(4)

Endrin ND(5) ND(5) ND(5) ND(5)

Cypermethrin ND(16) ND(16) ND(16) ND(16)

Permethrin ND(28) ND(28) ND(28) ND(28)

ND: non-detected at the detection limit mentioned; superscript value indicates thedetection limit (ng L�1).

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ponds and marshes present Eichhornia crassipes, Pontederia cor-data, Saggitaria montevidensis, Typha latifolia, Cortaderia selloana,Cyperus corymbosus, Salvinia biloba, and Pistia stratiotes.

2.3. Study species

L. limellum (Amphibia, Anura, Hylidae) was selected because itis frequently found in modified environments such as rice fields(Duré et al., 2008). It is widely distributed along Paraguay andParaná rivers ecosystems (Cei, 1980). In Argentina, this frog is ca-tegorized as ‘‘not threatened’’ (Vaira et al., 2012) and is distributedin the provinces of Buenos Aires, Formosa, Chaco, Corrientes,Santiago del Estero, Entre Ríos, and Santa Fe. L. limellum is a small-sized species, varying from 16 to 21 mm at adult stages, and it isusually associated with aquatic macrophytes (Peltzer et al., 2006;Sánchez et al., 2009).

2.4. Animal sampling

Forty seven adult males of L. limellum were collected by handfrom the sampling sites on two occasions: immediately (24 h;RS¼13 and RF¼12; 31 January 2013) and after 168 h (RS¼12 andRF¼10; 7 February 2013) aerial spraying with CPF. The two ob-servation periods were selected based on previous works (Sánchezet al., 1997; Lajmanovich et al., 2009; Attademo et al., 2011). All theanimals captured were males because they are easily identified inthe field by their calls and to minimize population impact (ASIHet al., 2001); gravid females were avoided. After capture, animalswere rapidly transported to the laboratory in darkened bucketscontaining water (approximately 2 cm deep) to minimize stress. Atthe laboratory, frogs were anaesthetized individually by immer-sion in a buffered solution of 0.1% tricaine methanesulfonate (MS-222), with approval of the animal ethics committee of the Facultyof Biochemistry and Biological Sciences, National University ofLittoral, and following the guidelines of ASIH et al. (2001). Eachindividual was weighed to the nearest 0.0001 g using a digitalbalance and their snout-vent length (SVL) was measured with adigital caliper. To compare the physiological condition of in-dividuals among populations, condition factor (CF) for each animalwas calculated and expressed as 100x[body weight (g)]/[length(cm)]3 (Bagenal and Tesch, 1978). Each individual was dissectedalong the mid-ventral line by making a longitudinal incision, anddigestive organs (stomach, intestine, and liver) were removed.Digestive organs contribute mainly to the OP detoxification (At-tademo et al., 2014b). Tissues were washed in distilled water andplaced on filter paper to remove excess fluids. Tissue samples werestored at �80 °C until biochemical analyzes were performed.

2.5. Water parameters

To analyze chlorpyrifos degradation (methyl chlorpyrifos, 99.5%and ethyl chlorpyrifos, 99.5%) in water from RF and RS, sampleswere collected in triplicate from a sprayed area after 24 and 168 hof pesticide application and simultaneously from the RS. Eachwater sample vas transported in a glass bottle to the laboratoryand either processed immediately or frozen at �18 °C.

In addition, other chemical pesticide screenings (Chem Service,Fluka and Applied Science, USA) were performed in water samplesin order to discard the presence of the following chemicals: γ-HCH(lindane) 99%; aldrin 99.9%; dieldrin 97.9%; p,p-DDT, o,p-DDT32and 65.6% p,p-DDE 99.5%; p,p-DDD 99%; heptaclor 99.2%; hepta-clor epoxide 99.5%; α and γ-Chlordane 99%; endosulfan I and II59.2 and 39.9%, endosulfan sufate 98.5%; endrin 99%; cypermehrin99%, and permethrin 99.5%. All pesticide residues were identifiedand quantified by VARIAN 3400 gas chromatograph (GC), withautomatic injector, electronic capture detector (ECD), DB-608

Megabore column (30 m�0.53 mm I.D., 1.5 mm), and DB-5 capil-lary column (30 m�0.25 mm i.d.) (J&W Scientific CA, USA), in-jector temperature 240 °C, detector temperature 320 °C and col-umn temperature 220 °C. For mass spectrometry, a Varian 2000chromatograph with mass selective detector (MSD) and an HP-5capillary column (30 m�0.25 mm ID) was used.

2.6. Enzymatic assays

Whole tissue samples were homogenized on ice in 20% (w/v)buffer containing 0.1% t-octylphenox- y polyethoxy ethanol (tri-tonX-100) in25mMtris (hydroxymethyl) amino methane hydro-chloride (pH 8.0) using a polytron. The homogenates were cen-trifuged at 10,000 rpm at 4 °C for15 min and the supernatant wascollected. AChE activity was determined colorimetrically followingEllman et al. (1961) procedure. The reaction mixture (final volume[F.V.¼930 ml]) consisted of 25 mM Tris–HCl containing 1 mMCaCl2 (pH¼7.6), 10 ml 20 mM acetylthiocholine iodide (AcSCh), and50 ml DTNB (3�10�4 M, final concentration). Variation in opticaldensity was measured in duplicate at 410 nm at 25 °C for 1 minusing a Jenway 6405 UV–vis spectrophotometer. Total Proteinconcentrations in the supernatants were determined using theBiuret method (Kingbley, 1942). AChE activity was expressed innmol of hydrolyzed substrate per minute per milligram of proteinusing a molar extinction coefficient of 13.6�103 M�1 cm�1. Car-boxylesterase was determined using α-naphthyl acetate (α-NA)substrate and specific enzyme activity was expressed asnmol min�1 mg�1 protein. The hydrolysis of α-NA by CbE wasmeasured as described by Gomori (1953) and adapted by Bunyanand Jennings (1968). The reaction medium (F.V.¼1950 ml) con-tained 25 mM Tris–HCl, 1 mM CaCl2 (pH¼7.6) and sample. Thereaction was initiated by adding 50 ml α-naphthyl acetate(1.04 mg ml�1 in acetone) after a preincubation period of 5 min at25 °C. The formation of naphthol was stopped after 10 min byadding 500 ml 2.5% sodium dodecyl sulfate and subsequently 0.1%of Fast Red ITR in 2.5% Triton X-100 in deionizer water (preparedimmediately before use). The samples were allowed to develop inthe dark for 30 min, and the absorbance of the complex was readat 530 nm (using a molar extinction coefficient of33.225�103 M�1 cm�1).

GST activity was determined spectrophotometrically using themethod described by Habig et al. (1974) and adapted by Habdouset al. (2002) for mammal serum GST activity. The enzyme assaywas performed at 340 nm in 100 mM Na–phosphate buffer (pH

Fig. 2. Comparative values of enzymatic activity in stomach of L. limellum. A: acetylcholinesterase (AChE). B: carboxylesterase (CbE). C: glutathione-S-transferase (GST) andD: catalase (CAT). Bars represent the mean7SEM. nPo0.05 compared with the reference site at 24 and 168 h.

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6.5) [F.V.¼920 ml]), 20 ml of 0.2 mM 1-chloro-2, 4-dinitrobenzene,50 ml of 5 mM reduced glutathione, and the sample. Enzyme ki-netics assays was performed at 25 °C and whole GST activity wasexpressed as mmol min�1 mg�1 protein using a molar extinctioncoefficient of 9.6�103 M�1 cm�1

CAT activity was measured using the method described by Aebi(1984), and was expressed as umol H2O2 mmol min�1 mg�1 pro-tein using a molar extinction coefficient of H2O2

40�10�3 L mol cm�1. The reaction medium was 50 mM phos-phate buffer (pH¼7.2) and 30 mM H2O2 and absorbance was readon the spectrophotometer at a wavelength of 240 nm at 25 °C(quartz cuvette).

2.7. Data analysis

Data of enzymatic activities were expressed as the mean andstandard error of the mean (x7SEM). The influence of samplingsites (RF and RS) and sampling periods (24 and 168 h) on AChE,CbE, CAT, and GST enzyme activities in tissue was analyzed withthe Student t-test. The Student t-test was also used for comparisonof CF. Data was tested for homogeneity and normality of variance(Kolmogorov–Smirnov test and Levene test). Statistical analyzeswere performed using INFOSTAT/ P 1.1 for Windows software(Grupo InfoStat Professional, FCA, Universidad Nacional de Cór-doba, Argentina). The criterion for significance was po0.05.

3. Results

3.1. Water parameters

Water concentrations of pesticides were below their respectivelimits of detection (Table 1). CPF was the only OP detected in watersamples from RF, at concentrations of 1388 ng L�1 at 24 h and97 ng L�1 at 168 h.

3.2. Condition factor

The mean condition factor (CF) values for L. limellum collectedfrom RF was 9.7170.56 at 24 h and 11.2270.70 at 168 h; thesevalues were similar to those recorded in frogs from RS:10.7070.52 at 24 h and 11.1470.43 at 168 h (Student t¼1.30,p40.05 and t¼1.01, p40.05).

3.3. B-esterase and oxidative stress enzymes in digestive tissues

3.3.1. StomachB-esterase (AChE and CbE) activity and oxidative stress (GST

and CAT) for each site (RF and RS) and two sampling periods (24and 168 h) are summarized in Figs. 2–4. In frog stomachs, AChEactivity was lower for individuals collected from RF than for thosefrom RS at 24 h (Student test t¼6.24, po0.001; Fig. 2A) and 168 h(t¼2.71, po0.05¼0.016; Fig. 2A). Reference CbE activities were38.8378.92 nmol min�1 mg�1 protein TP at 24 h and29.7972.86 nmol min�1 mg�1 protein at 168 h. CbE activity wassignificantly lower in individuals from RF than in those from RS at

Fig. 3. Comparative values of enzymatic activity in intestine of L. limellum. A: acetylcholinesterase (AChE). B: carboxylesterase (CbE). C: glutathione-S-transferase (GST) andD: catalase (CAT). Bars represent the mean7SEM. nPo0.05 compared with the reference site at 24 and 168 h.

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24 (t¼2.43, po0.05; Fig. 2B) and 168 h (t¼2.45, po0.05; Fig. 2B).GST activity in stomach tissue of RS individuals was 179.82714.87 nmol min�1 mg�1 protein at 24 h and 202.17733.74 nmol min�1 mg�1 protein at 168 h. GST activity exhibitedsignificantly lower values in individuals collected from RF than inthose from RS at 168 h (t¼2.21, po0.05; Fig. 2C). Similarly, CATactivity was significantly lower in individuals collected from RF at24 h (t¼9.14, po0.001; Fig. 2D) than in RS individuals.

3.3.2. IntestineMean values of the AChE activity in intestine were

12.2670.70 nmol min�1 mg�1 protein at 24 h and 10.47.70.54 nmol min�1 mg�1 protein at 168 h. AChE activity was sig-nificantly lower in individuals collected from RF at 24 h than in RSindividuals (t¼5.43, po0.001; Fig. 3A). Control CbE activity in RS in-dividuals was 58.2773.00 nmol min�1 mg�1 protein at 24 h and65.9873.01 nmol min�1 mg�1 protein at 168 h. CbE activity wasdifferent between RS and RF at 24 h (t¼2.12, po0.05; Fig. 3B). GSTand CAT activity in the intestine of individuals from RF was264.77759.08 nmol min�1 mg�1 protein and 91.3576.01 nmolmin�1 mg�1 protein at 24 h; and 308.70740.18 nmol min�1 mg�1

protein and 79.4577.31 nmol min�1 mg�1 protein at 168 h. GST andCAT activities were significantly lower in individuals collected from RFthan in those from RS at 24 h (t¼2.39, po0.05 and t¼6.72, po0.001;respectively), but only CAT was significantly lower in RF individualsthan in RS individuals at 168 h (t¼3.82, po0.001; Fig. 3C and D).

3.3.3. LiverMean values of the AChE activity were 16.0571.20 nmol

min�1 mg�1 protein at 24 h and 13.9370.84 nmol min�1 mg�1

protein at 168 h in RS. AChE activity was significantly lower in in-dividuals collected from RF at 24 h (t¼4.16, po0.001) and 168 h(t¼2.48, p¼0.026; Fig. 4A) than in RS individuals. Control CbE activityin RS was 127.4879.92 nmol min�1 mg�1 protein at 24 h and107.5879.71 nmol min�1 mg�1 protein at 168 h. CbE activity wasdifferent between individuals from RS and RF at 24 h (t¼3.98,po0.001) and 168 h (t¼4.92, po0.001; Fig. 4B). GST and CAT activityin liver of individuals from RF was 470.78740.51 nmol min�1 mg�1

protein and 199.2479.55 nmol min�1 mg�1 protein at 24 h; and515.77732.21 nmol min�1 mg�1 protein and 202.9375.12 nmolmin�1 mg�1 protein at 168 h. GST and CAT activities were sig-nificantly lower in individuals collected from RF than in those from RSat 168 h (t¼9.64, po0.001 and t¼3.94, po0.001; respectively Fig. 4Cand D).

4. Discussion

Chlorpyrifos (CPF) was applied to control numerous pests inrice agroecosystems during the evaluated crop season. A notabledegradation (412-fold) of CPF over seven days was found in waterof the RF. Similar concentrations were reported for Central Cali-fornia Coast (Hunt et al., 2003). Two separate studies agreed thattransformation of CPF is rapid during the first few days followingapplication, with this transformation being generally attributed topotential volatilization and degradation (Racke, 1992).

A significant reduction of AChE and CbE activities was detectedin L. limellum from RF in stomach and liver at 24 and 168 h, and inintestine only at 24 h with respect to RS individuals.

Studies exploring variation of AChE and CbE in tissue samples

Fig. 4. Comparative values of enzymatic activity in liver of L. limellum. A: acetylcholinesterase (AChE). B: carboxylesterase (CbE). C: glutathione-S-transferase (GST) and D:catalase (CAT). Bars represent the mean7SEM. nPo0.05 compared with the reference site at 24 and 168 h.

A. Maximiliano Attademo et al. / Ecotoxicology and Environmental Safety 113 (2015) 287–294292

of adult frogs are scarce. However, previous experiments in ourlaboratory showed similar results in another native species (Hyp-siboas pulchellus) at tadpole stages, as well as in fish (Synbranchusmarmoratus) exposed to fenitrotion (OP) (Junges et al., 2010). Si-milarly, Colombo et al. (2005) and Widder and Bidwell (2008)observed a significant reduction of cholinesterase (AChE) activity inanuran tadpoles (Xenopus laevis, Hyla chrysoscelis, Rana sphenocephala,Acris crepitans, and Gastrophryne olivacea) exposed to CPF. This enzymeinhibition in tissues by OP may persist for days to weeks (Morganet al., 1990; Wijeyaratne and Pathiratne, 2006), and is in accordancewith the present observation for B-esterases after 168 h of application.Similarly, Strauss and Chambers (1995) found that CPF was a powerfulinhibitor of AChE activity in fingerling channel catfish (Ictaluruspunctatus). After exposure to AChE inhibitors, the length of AChE de-pression affects the recovery of organisms and their possibility ofsurvival under field conditions (Beauvais et al., 2001), which is con-sistent with the present results. Wijeyaratne and Pathiratne (2006)observed significant inhibition of brain AChE activity in fish (Rasboracaverii) from rice fields, with this inhibited activity persisting even 65days after insecticide application. Among B-esterases, CbEs are im-portant to reduce OP toxicity (Stenersen, 2004) and these isozymesmay contribute to pesticide tolerance due to their capability to bind toOPs, decreasing the effective concentration of the pesticide (Wheelocket al., 2008). Some environmental pesticides found to inhibit AChEactivity may also inhibit CbE activity (Küster, 2005); here we foundlower CbE activities in all digestive tissues of L. limellum individualsfrom RF.

Moreover, GST activity decreased in intestine of L. limellumfrom the RF at 24 h and stomach and liver at 168 h. This enzyme

catalyzes the conjugation of electrophilic xenobiotics to GSH(Gadagbui and James, 2000), plays an important role in protectingtissues from oxidative stress (Ognjanovic´ et al. 2003; Ferrari et al.2011) and its inhibition could lead to an imbalance of the cellularredox status. Likewise, Frasco and Guilhermino (2002) and Özkolet al. (2012) demonstrated that dimethoate (OP) inhibits GST ac-tivity in fish (Poecilia reticulata) and in different tissues of frogs(Rana ridibunda). Accordingly, a lower level of GST activity has alsobeen reported at 48 and 96 h in congeneric amphibian tadpoles(Scinax squalirostris) in rice crops of an agroecosystem (Attademoet al., 2014a).

In addition, CAT activity decreased in stomach and liver of L.limellum frogs at 168 h, and only in intestine did it decrease after24 and 168 h in individuals from RF. A reduction in CAT activitymay be caused by the flux of superoxide radicals (O2�) induced bythe CPF (Ahmad et al., 2000). Likewise, Wielgomas and Krechniak(2007) reported the alteration in CAT activity in wistar rat (species)liver after exposure to CPF. Mekail and Sharafaddin (2009) foundthat CAT activity decreased in brain, liver and kidney of weanlingrat treated with OP (diazinon). More recently, Özkol et al. (2012)reported a decrease in CAT activity in tongue of frogs (R. ridibunda)exposed to omethoate (OP). The use of GST and CAT enzymes inadult frogs exposed to CPF reinforces a rational use of biomarkersof oxidative stress in biomonitoring of pesticides.

5. Conclusion

Overall, our results demonstrate (1) CFP applications in ricefield produces mortality of L. limellum adults, (2) inhibition of

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AChE and CbE at 24 and 168 h in all digestive tissues in individualsfrom the rice field, (3) a decrease of GST and CAT activities instomach and liver of L. limellum individuals at 168 h, and in in-testine at 24 and 168 h after CPF application. The demonstratedtoxicity in L. limellum at biochemical level is alarming and a po-tential negative effect of pesticide exposure on sympatric amphi-bian species seems likely. The use of enzyme activities as bio-markers are of great importance in understanding the impacts ofpesticides on amphibian that occur in rice agroecosystems and inestablishing safe limits on the use of these products in these en-vironments. Based upon our results, it can be concluded thatbiomarker responses in anurans from rice agroecosystems is veryuseful for providing lines of evidences that can contribute to en-vironmental monitoring programs designed for various aspects ofenvironmental risk assessment (ERA).

Acknowledgements

We thank the field owner for allowing us to conduct the study.We also thank J. Brasca English Editing Service and A. Bassó forfield assistance. This study was supported in part by Consejo Na-cional de Investigaciones Científicas y Técnicas (CONICET), AgenciaNacional de Promoción Científica y Tecnológica (ANCyT) and Cursode Acción para la Investigación y Desarrollo (CAIþD-UNL).

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